Mutant bank

ABSTRACT

The invention relates to a mutant bank of diploid micro-organisms which consists of a population of mutant cells in which at least one cell has a random mutation which disrupts the activity of at least one gene, wherein the micro-organism is inducible into haploid form. The invention further relates to a method of using the mutant bank to identify the genes which contribute to a chosen phenotype.

The present invention relates to the generation of mutant banks ofmicro-organisms comprising a population of mutant cells in whichessentially every gene within the genome is inactivated and usesthereof.

Due to the advances made by various genome sequencing projects, the arthas reached the position that many genes have now been cloned andsequenced. However, a problem remains that for most identified genes alink has not been made with function (i.e. how an identified gene mayeffect phenotype).

One way by which gene function may be identified is to compare thephenotypes of mutant cells or organisms with the known phenotype of a“wild-type” cell or organism. The gene responsible for this phenotypicchange may be identified by DNA sequencing of mutant and wild-typecells. The identity of the gene product may then be easily established.Such knowledge can give rise to industrially useful processes in whichthe activity of the gene, or its product, is modulated to achievespecific goals. For instance, identification of a mutation that reducesthe growth of a pathogenic mould provides valuable information fordeveloping therapeutic strategies.

Various strategies have been proposed for identifying mutations inspecific organisms. For instance, an insertional mutation technique forplants is disclosed in WO 99/14373. This document discloses a means ofselecting and identifying insertional mutations in a population of plantcells based upon a method utilising the Ti plasmid of Agrobacteriumtumefaciens (a gram-negative soil bacterium) for effectingtransformation.

EP-A-0 870 835 also relates to the use of the Ti plasmid ofAgrobacterium. It discloses that the abovementioned Ti plasmid techniquemay also be used to transform moulds from the fungal subdivisionsAscomycotina, Basidiomycotina, Deuteromycotina, Mastigomycotina andZygomycotina.

One of the problems associated with the mutant populations disclosed bythe prior art is that often no account is taken of the effect of lethalmutations. A lethal mutation results in the death of particularindividuals and therefore any mutant population generated according tosuch prior art techniques would result in an “incomplete” populationcomprising “non-lethal” mutants. It is an aim of the present inventionto obviate or mitigate this disadvantage, as well as others, associatedwith the prior art.

According to a first aspect of the present invention there is provided amutant bank of micro-organisms comprising a population of mutant cellsin which at least one cell has a mutation that disrupts the activity ofat least one gene, said micro-organisms being diploid and beinginducible into haploid form.

The micro-organisms may normally occur in a haploid form and are,preferably, first induced into the diploid form.

Preferably, a plurality of cells in the population each individuallyhave a mutation which disrupts the activity of at least one gene, sothat, preferably, collectively the said plurality of cells havemutations in a plurality of genes within the genome.

Preferably, the said plurality of genes makes up 0.001% of the geneswithin the genome, more preferably 0.01%, most preferably 0.1%, evenmore preferably, 10%, even more especially substantially all theessential genes and most especially all the genes of the genome of themicro-organism.

According to a second aspect of the present invention there is provideda method of generating a mutant bank of micro-organisms comprising apopulation of mutant cells in which at least one cell has a mutationthat disrupts the activity of at least one gene, said method comprisingthe steps of:

(i) culturing a population of micro-organisms; and

(ii) inducing a mutation in at least one cell of the population whichmutation disrupts the activity of at least one gene in the genome of themicro-organism, said micro-organisms being diploid and being inducibleinto haploid form.

Preferably, the method comprises the further step of exposing thediploid micro-organisms to an agent that induces the micro-organismsinto haploid form after generating the mutant bank.

Preferably, the method comprises the further step of separating andculturing the haploid micro-organisms as single clones after theinduction of the micro-organisms into haploid form.

Preferably, the method comprises the further step of selecting clonesfor which the chosen phenotype is altered relative to a wild typemicro-organism after separating and culturing the haploidmicro-organisms.

Preferably, the method comprises the further step of identifying themutated gene in each of the selected clones after selecting clones forwhich the chosen phenotype is altered relative to a wild typemicro-organism.

The micro-organisms may normally occur in a haploid form. Therefore,preferably, the method of the second aspect further includes the step offirst inducing the haploid into diploid form prior to culturing the saidpopulation.

According to a third aspect of the present invention there is provided amethod of identifying genes in a micro-organism which contribute to achosen phenotype comprising:

(i) generating a mutant bank of diploid micro-organisms consisting of apopulation of mutant cells in which at least one cell has a mutationwhich disrupts the activity of at least one gene;

(ii) exposing the diploid micro-organisms to an agent that induces themicro-organisms into haploid form;

(iii) separating and culturing the haploid micro-organisms as singleclones; and

(iv) selecting clones for which the chosen phenotype is altered relativeto a wild type micro-organism; and

(v) identifying the mutated gene in each of the selected clones.

According to a fourth aspect of the present invention there is provideda mutant bank of diploid micro-organisms consisting of a population ofmutant cells in which each individual cell has a mutation that disruptsthe activity of one gene, said population collectively having a mutationin every gene within the genome and wherein the mutant bank may beinduced into haploid form.

According to a fifth aspect of the present invention there is provided amethod of identifying genes in a micro-organism which contribute to achosen phenotype comprising:

(i) generating a mutant bank of diploid micro-organisms consisting of apopulation of mutant cells in which each individual cell has a mutationwhich disrupts the activity of one gene, said population collectivelyhaving a mutation in every gene within the genome;(ii) exposing the diploid micro-organisms to an agent that induces themicro-organisms into haploid form;(iii) separating and culturing the haploid micro-organisms as singleclones; and(iv) selecting clones for which the chosen phenotype is altered relativeto a wild type micro-organism; and(v) identifying the mutated gene in each of the selected clones.

By “disrupts the activity of one gene” we mean that the gene contains amutation that prevents transcription or translation of the proteinencoded by the gene.

Alternatively, the translated protein has no activity, or at leastaltered activity, relative to the wild type gene product that results ina measurable phenotypic change.

The mutant banks may be used according to the present invention toscreen the genome of the micro-organism for genes that are linked with aspecific phenotype. This knowledge may be used to develop modulators ofthe gene and may lead to the development of new medicaments, pesticides,disinfectants etc. which may be targeted against the micro-organism.

The prior art does not contemplate mutant banks of micro-organismscomprising a population of mutant cells in which essentially theactivity of every gene within the genome is disrupted and which may beconverted between diploid and haploid forms.

One of the advantages of the mutant banks according to the invention isthat a complete diploid population may be cultured under suitableconditions. This is possible because the wild type copy of the gene willrescue any individual that may otherwise grow poorly, or even not beviable, should the mutant copy of the gene predominate. Thus, thediploid mutant bank may comprise a population of mutant, viable cells inwhich essentially the activity of every gene within the genome isdisrupted rather than an “incomplete” population comprising “non-lethal”mutants.

Furthermore, the mutant bank may be comprised within a population ofcells which also comprises cells in which no mutations may have occurredand/or in which more than one mutation may have occurred.

When the diploid mutant bank is induced into haploid form (step (ii)) ofthe third and fifth aspects any phenotypic changes in the haploidmutants may be interpreted by a technician to provide valuableinformation about gene function. For instance, the haploid cells may begrown under normal culture conditions. Under these circumstances, thedeath of a haploid clone would imply that the mutation caused a lethalphenotypic change. The clone could then be isolated from the originaldiploid sample and the mutant gene identified.

When organisms are used which are naturally found as haploids, it ispreferred that the method of the third and fifth aspects of theinvention comprises an initial step of converting the haploid cells intodiploid form before mutations are induced as defined by step (i) of themethod of the third and fifth aspects of the invention.

Organisms which are normally haploid, may be induced into diploid formutilising the methods described in the Examples at 1.4 and 1.5.

It is preferred that the mutant banks are used according to theinvention to screen the genome of a micro-organism for genes whichcontribute to a chosen phenotype by growing the micro-organisms inpermissive or selective growth media. The choice of permissive orselective growth media will depend upon the phenotype of interest. Forinstance, the haploid cells may be initially grown in a media which isosmotically buffered such that cells with weak cell walls (which wouldlyse in a normal media) are able to survive. Clones which survive undersuch circumstances, and which do not when grown in normal media, may beselected. The mutant genes, (and thereby the gene products), in theselected clones may be identified by conventional molecular biologytechniques. The identification of such gene products may be used as anaid for rationally identifying gene products which regulateosmoregulation and could also identify leads for developinganti-microbial agents that will induce cell lysis by disruptingosmoregulation.

Mutant banks used according to the invention are particularly useful foridentifying new anti-microbial drug targets. By a “drug target” we meanthe site of action of a drug.

By identifying essential genes in the mutant banks and then analyzingthem (e.g. using computer analysis—bioinformatics), the commonality ofthe gene in other micro-organisms (or fungi) can be established and anysignificant differences between mammals (e.g. man or mouse) and themicro-organism defined. For instance, a good antifungal drug targetwould be a protein encoded by a gene which is shared by all fungi (andbe very similar between different species) and not found in man at all.By a continuous process of identifying possible new targets, on thebasis that an identified mutant gene had an effect on phenotype, andthen bioinformatic analysis to rule candidates out, multiple new targetsmay be identified in a short time-frame.

The mutant banks also provide a simple means of establishing themechanism of action of a compound known to have anti-fungal activity.Colonies from the banks may be plated out on media containing theanti-fungal compound of interest and resistant or less susceptiblecolonies examined to determine which gene has been inactivated byinsertional mutagenesis. This gene product is the likely target for thatanti-fungal agent. This represents a rapid and simple means fordetermining the mechanism-of-action of the agent.

In addition to identifying and validating anti-fungal targets, otherspecial characteristics can be selected for. For instance, thetransporter genes that mediate drug resistance may be identifiedaccording to the method of the invention. There are about 30 transportergenes in yeast but only 3 have been found in Aspergillus fumigatus todate. In fungi the most common resistance mechanism is due to the activeexport of potentially lethal drugs from within the fungal cell.Transport mutants may be identified by selecting from the haploid mutantbank for drug sensitive strains (indicating that a transporter gene isnon-functional). Drug sensitive mutants (i.e. incapable of drug export)will make good strains for drug development because the potential forfuture resistance by these efflux mechanisms can be directly assessed.

The micro-organism may be a yeast or any fungus, preferably, afilamentous fungus which may be switched between diploid and haploidform.

A most preferred micro-organism is A. fumigatus and related strains. A.fumigatus is a haploid organism and the most common human mouldpathogen. The genome of A. fumigatus is thought to comprise 8,000-11,000genes. It is important when developing anti-microbial agents to select asuitable target which will not only kill the micro-organism but whichare also selective. In this respect it is expected that approximately750-1000 genes in A. fumigatus will be essential and therefore potentialtargets. Of the essential genes possibly a quarter will be new geneswith no known counterpart in yeast-fungi, over 100 are likely to bestructural genes (which do not make good targets) and as many as 500will be common to man. This leaves an estimated 150 candidate targetswhich may encode proteins that could represent leads for developinganti-microbial agents. The method of the second aspect of the inventionmay be used to identify candidate clones. Further screening of thecandidates may be achieved by employing bioinformatics and it isestimated that such an approach will generate 15-25 validated targets.Some may be shared by bacteria, raising the possibility of findinganti-microbials that have both antibacterial and antifungal activity.

A. fumigatus clinical isolates AF300 and AF293 (available to the publicfrom the NCPF repository (Bristol, U.K.) and the CBS repository(Belgium)) are preferred strains which may be formed into mutant banksaccording to the present invention.

Another preferred micro-organism is Candida glabrata. Candida speciesare important yeast fungal pathogens of humans. Until recently, C.glabrata was considered a relatively non-pathogenic commensal fungalorganism of human mucosal tissues. However, with the increased use ofimmunosuppressive agents, mucosal and systemic infections caused by C.glabrata have increased significantly, especially in the humanpopulation infected with HIV. C. glabrata currently ranks second orthird as the causative agent of superficial (oral, oesophageal, vaginal,or urinary) or systemic candidal infections. A number of factors havebeen proposed as being important to virulence but details of thehost-pathogen interaction are, however, largely unknown. A majorobstacle in C. glabrata infections is their innate resistance to azoleantimycotic therapy, which is very effective in treating infectionscaused by other Candida species. C. glabrata, formerly known asTorulopsis glabrata, contrasts with other Candida species in itsnondimorphic blastoconidial morphology and haploid genome. We haverealised that the possession of a haploid genome is a useful feature inthe production of mutant banks as most Candida species are obligatediploids with no known haploid forms and therefore C. glabrata is apreferred micro-organism which may be made into a mutant bank accordingto the invention.

A. fumigatus and C. glabrata normally have only one set of chromosomes(haploid) and conventionally would not be considered for the formationof a bank of mutants because lethal gene knockouts would not survive andtherefore the bank would be incomplete. However, the inventors haveappreciated that these moulds can be induced to carry a double set(diploid) of genes. They therefore realised that this property could beexploited such that a stock mutant bank may be maintained in diploidform. One gene from each pair should be in wild type form and therebyallow the organisms to be phenotypically normal. When desired the cellsmay be allowed to return to haploid form and the effects of the mutantgene identified.

It will be appreciated that the method of the invention used in A.fumigatus may be usefully employed to gain knowledge relating tohomologous genes for other species of micro-organism. For instance,biotechnology companies are interested in improving strains offilamentous fungi which are used extensively in industry for theproduction of soy sauce, citric acid etc. Furthermore, the production ofmutant banks or knowledge gained from the Aspergillus mutant bank inplant pathogenic fungi is useful to the agrochemical industry for thedevelopment of broad-specificity anti-fungal drugs.

Preferably, the mutant banks comprise a population of cells in which themutations have been randomly induced. Preferably, there has been arandom knock out of essentially all genes in the micro-organism.

The mutant banks may be generated (according to step (i) of the thirdand fifth aspects of the invention) by exposing a population ofmicro-organisms to an agent which randomly causes single mutations ineach individual micro-organism. It is preferred that the mutant bank isformed by an insertional mutagenesis method. Preferably, a DNA moleculeis inserted into each gene to cause the mutation. The DNA molecule maybe a selectable marker which is, preferably, a pyrG gene, morepreferably, the pyrG gene from A. fumigatus.

Advantageously, the pyrG gene is a preferred selectable marker becauseit may be very tightly regulated since there is an absolute requirementof pyrG mutants for uridine and uracil supplementation in the growthmedium. Advantageously, this substantially reduces the number of falsenegatives.

Preferably, the pyrG gene is harboured on a plasmid which is,preferably, an Aspergillus pyrG containing plasmid.

The micro-organism may be a fungus, preferably, a filamentous fungusand, most preferably, A. fumigatus. In a preferred embodiment themicro-organism is AF300 or AF293.

Alternatively, the micro-organism may be a yeast, preferably, C.glabrata.

Preferably, the mutations have been randomly induced, more preferably,by an insertional mutagenesis method. Preferably, a DNA molecule isinserted into each gene to cause the mutation. The DNA molecule may be aselectable marker which is, preferably, a pyrG gene, more preferably,the pyrG gene from A. fumigatus. Preferably, the pyrG gene is harbouredon a plasmid which is, preferably, an Aspergillus pyrG containingplasmid.

Examples of the pyrG containing plasmid are shown in FIGS. 6, 7, 10 and11.

Preferably, the plasmid is introduced into the micro-organism byelectroporation (see section 1.7.1).

Preferably, the diploid micro-organism is a mutant for the pyrG⁻phenotype.

Advantageously, this allows for the selection of the pyrG genecontaining plasmids.

Preferably, the diploid mutant micro-organism is generated using thefollowing steps:

(i) isolating a first haploid mutant comprising a first auxotrophicmarker wherein the first auxotrophic marker results in the pyrG⁻phenotype;

(ii) isolating a second haploid mutant using the first haploid mutantisolated in step (i), said second mutant comprising the first pyrG⁻auxotrophic marker and a second auxotrophic marker;

(iii) isolating a third haploid mutant using the first haploid mutantisolated in step (i), said third mutant comprising the first pyrG⁻auxotrophic marker and a third auxotrophic marker; and

(iv) mating the second and third haploid mutants isolated in steps (ii)and (iii) to generate the diploid mutant exhibiting the pyrG phenotype.

Preferably, the second and third auxotrophic markers are selected fromniaD⁻ or cnx⁻. Preferably, cells which are unable to utilise nitratealone were classified as niaD⁻ mutants and, preferably, mutants unableto utilise mitrate or hypoxanthine were classified as cnx⁻ mutants.

Preferably, the mutant diploid micro-organism is a double mutant for thepyrG⁻ phenotype, i.e. pyrG⁻/pyrG⁻ phenotype. Preferably, the doublemutants (pyrG⁻/pyrG⁻ phenotype) were made using a haploid single pyrG⁻mutant as a parental strain.

Preferably, the order of making the diploid double mutants was found tobe important since starting with a niaD⁻ or cnx⁻ mutant as the parentalstrain we were unable to then isolate a niaD⁻/pyrG⁻ or cnx⁻/pyrG⁻mutant.

Preferably, the method comprises use of the Frontier method (section:1.4.1) for the production of diploid micro-organisms in A. fumigatus.

Preferably, the haploid mutants are incubated at a temperature in therange of 20-36° C., more preferably, in the range of 24-33° C. and, mostpreferably, in the range of 26-30° C. Most preferably, the haploidmutants are incubated at 28° C.

Advantageously, incubation at a lower temperature slows down growth ofthe haploid mutants thereby increasing the chance of heterokaryonformation and the subsequent production of diploid mutants with which tomake the mutant bank. Preferably, putative mutant cell transformants areidentified using a microscope when they are substantially not visible tothe naked eye at the earliest stage possible in their development.Preferably, putative mutant cell transformants are identified at 32 to48 hours in to their development, more preferably, at 34 to 46 hoursand, most preferably, at 36 to 44 hours in to their development.

Preferably, the minimum feasible time that putative mutant celltransformants could be identified using a microscope is 36-48 hrs. Mostmethods rely on the macroscopic identification of colonies, i.e. by eyewhen they have reached a particular size. By this time, many of thecolonies are sporulating which poses a cross-contamination problem withother nearby putative transformants. More importantly, due to therandomness of the transformation procedure, there will be a whole rangeof transformants which are affected to a greater or lesser degree intheir growth rates. Some of these transformants may never reachmacroscopic size and would hence be lost from the population of themutant bank as a whole. Identification of these transformants bymicroscope at an early stage allows them to be transferred to a richer,more complete medium where they have a better chance to grow to a sizewhere DNA can be extracted and the genetic mutation identified.

According to a still further aspect of the present invention there isprovided SEQ ID No. 27 and homologues thereof, preferably, functionaland/or structural homologues thereof.

SEQ ID No. 27 (see sequence listing) may be used for the manufacture ofa medicament for treatment of an infection of A. fumigatus.

In an alternative emodiment, the insertional mutagenesis may be carriedout using the Ti plasmid. The use of the Ti plasmid in filamentous fungihas previously been described (de Groot et al. (1998) NatureBiotechnology 16:839-842), but it has not been used for the generationof mutant banks according to the first aspect of the invention nor hasit been used in filamentous fungi such as A. fumigatus or in C.glabrata.

The Ti plasmid transformation methods disclosed in EP-A-0 870 835(incorporated herein by reference) may be used, and adapted asappropriate, to generate mutant banks according to the invention.According to one embodiment (see the example), the physical steps oftransforming diploid A. fumigatus may be essentially the same as thosedisclosed in EP-A-0 870 835.

The mutant bank may be generated by transformation with a Ti plasmidvector based on the binary vector pBIN19 (disclosed in: Bevan. (1984)Nucleic Acids Research 22: 8711-8721).

It is most preferred that LBA4404 and GV3101 strains of A. tumefaciens(identified in Table 1) are used to generate the mutant banks of thepresent invention.

TABLE 1 Reference Strain Genotype disclosing strain LBA 4404 Ach5 Rif^(r) containing plasmid Oooms et al. (1981) pAL4404 (ΔT_(L), ΔT_(R),Δtra, Δocc) a Gene 14: 33-50. deletion of pTiAch5. GV3101 C58C1 Rif ^(r)containing plasmid Koncz & Schell, (1986) pMP90 (Gm ^(r)) a deletion ofMolecular and General pTiC58. Genetics 204: 383-396.

Most preferred transformation steps are described in 1.7 of the Example.

Following generation of the diploid mutant bank, the cells are inducedinto haploid form according to step (ii) of the method of the invention.A preferred technique for inducing the cells into haploid form isdescribed in the Example under 1.6.

The haploid cells from step (ii) are then separated and culturedaccording to step (iii). This may be achieved by conventional dilutionand spread plating of the culture and is also described at 1.6 of theExample.

Clones with an altered phenotype (step (iv)) may be isolated by avariety of conventional means (e.g. by growth on a selective medium).

Once clones have been selected according to step (iv) of the method ofthe invention it is preferred that the mutated gene is identified.Identification of the mutated gene may be achieved by including sectionsof marker DNA during the generation of the mutant bank. For instance, agene may be insertionally inactivated by incorporating a marker DNAsequence which may be later identified using conventional molecularbiology techniques (e.g. using labelled probes for the marker).

Alternatively, the marker may be used as a target for a primer which canbe used to directly sequence and amplify the mutant gene. This approachhas several advantages over known mutational techniques. For instance:

(1) it is not necessary to have discovered the gene previously;

(2) the essential function of the gene is established concurrently withits identification; and

(3) it is extremely rapid.

Various methods known to those skilled in the art may be used toidentify the gene. Preferred methods are outlined at 1.9 and 1.10 of themethods section of the Example.

All of the features described herein may be combined with any of theabove aspects, in any combination.

An embodiment of the present invention will now be described, by way ofexample, with reference to the accompanying drawings in which:

FIG. 1 is a schematic representation of plasmid pAN7-1;

FIG. 2 is a schematic representation of plasmid pRok2;

FIG. 3 is a schematic representation of plasmid pRic1;

FIG. 4 is a schematic representation of plasmid pUC 18;

FIG. 5 is a schematic representation of plasmid pRic2;

FIG. 6 is a schematic representation of plasmid pRic3;

FIG. 7 is a schematic representation of plasmid ppyrG;

FIG. 8 is a schematic representation of plasmid pCR2.1;

FIG. 9 is a schematic representation of plasmid pTT;

FIG. 10 is a schematic representation of plasmid pMB2;

FIG. 11 is a schematic representation of plasmid pMB3;

FIG. 12 is a BLASTX search result used for the identification of mutatedgene in strain MA205;

FIG. 13 is a Southern blot showing integration events; and

To the Sequence Listing.

EXAMPLE

1. Methods

1.1 Bacterial and Fungal Strains:

For bacterial cloning the E. coli strain Top10 (F⁻, mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZAM15 ΔlacX74 deoR recA1 araD139 Δ(ara-leu)7697galU galK rpsL(Str^(R)) endA1 nupG) was used.

The A. tumefaciens strains LBA4404 and GV3101 (see Table 1) were usedfor the transformation of A. fumigatus.

A. fumigatus clinical isolates AF300 and AF293 (available to the publicfrom the NCPF repository (Bristol, U.K.); the CBS repository (Belgium)or from Dr. David Denning clinical isolate culture collection, HopeHospital, Salford. U.K.) are preferred strains which may be formed intomutant banks according to the present invention.

AF300: Isolated 1995. Royal Manchester Children's Hospital. Leukaemiapatient.

AF293: Isolated 1993. Donated by Shrewsbury PHLS. Lung biopsy, invasiveaspergillosis with aplastic anaemia.

1.2 Plasmid Construction:

1.2.1 Plasmid pRic1

Plasmid pRic1 (see FIG. 3) was constructed by cloning a 4 kbHindIII-BglII fragment, which is present on the vector pAN7-1 (Punt etal. (1987) Gene 56: 117-124; see FIG. 1) and contains the promoter fromthe Aspergillus nidulans gpd gene fused to the coding region of the E.coli hygromycin B phosphotransferase (hph) gene and followed byterminator sequences from the A. nidulans trpC gene, into theHindIII-BamHI site of the Cauliflower Mosaic Virus (CaMV) 35s promoterregion of the binary vector pRok2 (a derivative of the binary vectorpBinl9) pRok2 is disclosed in: Baulcombe et al. (1986) Nature 321:446-449; and FIG. 2).

1.2.2 Plasmid pRic2

Plasmid pRic2 (see FIG. 5) was constructed by cloning the entire pUC18plasmid (see: Yanisch-Perron et al (1985) Gene 33: 103-119; and FIG. 4)linearised with HindIII into the HindIII site of pRic1.

1.2.3. Plasmid pRic3

Plasmid pRic 3 (see FIG. 6) was constructed by cloning the entire ppyrGplasmid (Fungal Genetics Stock Centre, and FIG. 7) linearised with Xbalinto the Xbal site of pRok2.

The A. tumefaciens strain GV3101 was electroporated with the constructspRic1, pRic2 and pRic3 using a Biorad Genepulser (2.5 kV, 600 Ω, 25°F.). The constructs pRic1, pRic2 and pRic3 were transformed into A.tumefaciens strain LBA4404 using a triparental mating method as follows.Freshly grown cultures of E. coli Top10 containing pric1, pRic2 orpRic3, E. coli Top10 containing the helper plasmid pRK2013 and A.tumefaciens LBA4404 were mixed in the middle and streaked to the edgesof a LB plate which was then incubated overnight at 25° C. A loop ofcells from the LB plate was then streaked onto a fresh LB agar platecontaining rifampicin (200 μg/ml) and kanamycin (50 μg/ml) for pRic1transformations and rifampicin (200 μg/ml), kanamycin (50 μg/ml) andampicillin (100 μg/ml) for pRic2 and pRic3 transformations. This platewas incubated at 25° C. until single colonies started to form (thesesingle colonies represent A. tumefaciens LBA4404 containing pRic1, pRic2or pRic3).

1.2.4 Plasmids pMB2 and pMB3

The homologous pyrG gene from wild type AF293 was amplified usingprimers designed to the start of the promoter and end of the terminatorof the A. fumigatus pyrG gene (Weidener, G. and d'Enfert, C. (1998)Current Genetics 33, 378-385).

Primers AFpyrG5 (5′-cta cct cga gaa ttc gcc tca aac-3′; SEQ ID No 25)and AFpyrG3 (5′-ggc gac gaa ttc tgt ctg aga g-3′; SEQ ID No 26) wereused in PCR reactions containing Taq polymerase (ABGene) and Expand Pfupolymerase (Roche Molecular Systems).

Reactions which amplified the predicted 1.9 kb fragment were cloneddirectly. Where Taq polymerase was used, fragments were cloned directlyinto PCR cloning vector pTT (Genpak Ltd, see FIG. 8). Where Expand Pfuwas used, fragments were first treated with Taq polymerase for 10 min at72° C. Reactions were then passed through a cleanup column (Qaigen Ltd)to remove residual enzymes and nucleotides and then cloned into vectorpCR2.1 (Invitrogen Ltd, see FIG. 9) according to the manufacturersinstructions.

In both cases ligated vector/insert were transformed into Top10electrocompetent cells (Invitrogen Ltd) and plated onto LB agarcontaining ampicillin (100 μg ml). Following overnight incubation at 37°C. six individual colonies from each reaction were sub-cultured into LBbroth containing ampicillin (100 μg ml). After overnight incubation at37° C., plasmids were extracted using Qaigen Spin Mini plasmidextraction kits and digested with EcoRI.

Plasmids containing the predicted 1.9 kb insert released by EcoRIdigestion were deemed positive clones. Aliquots of selected positiveclones were sequenced to confirm the presence of the A. fumigatus pyrGgene. Sequencing was performed by MWG Biotech UK Ltd (Waterside House,Peartree Bridge, Milton Keynes, MK63BY).

Plasmid pMB2 (see FIG. 10) was generated from a Taq polymerase cloneinserted into pTT. Plasmid pMB3 (see FIG. 11) was generated from a Pfupolymerase clone inserted into pCR2.1.

1.3 Isolation of A. fumigatus Haploid Auxotrophs

1.3.1 Nitrate Assimilation Mutants (niaD⁻ and cnx⁻)

Haploid strains of A. fumigatus AF293 were identified which weredeficient in the utilisation of nitrogen in the form of nitrate. Nitratemutants were selected after inoculation of AF293 conidia (10⁷ per plate)on chlorate plates (600 mM sodium chlorate) supplemented with 10 mMasparagine. Colonies growing on chlorate plates were characterisedfurther by their ability to grow on agar containing either 10 mMnitrate, 10 mM nitrite or 10 mM hypoxanthine as the sole nitrogensource. Colonies unable to utilise nitrate alone were classified asniaD⁻ mutants. Colonies unable to utilise nitrate or hypoxanthine wereclassified as cnx⁻ mutants. Reversion rates were checked by platingconidia onto nitrate media at 10⁷ conidia/ml. No reversion was seen atthis level.

1.3.2 pyrG⁻ Mutants

The pyrG⁻ phenotype is due to a mutation in the gene encoding orotidine5-phosphate decarboxylase. This causes a requirement for supplementationof the growth media with 10 mM uracil and 10 mM uridine in order toallow pyrG⁻ mutants to grow.

Generation of pyrG⁻ mutants is achieved by the use of the metabolicinhibitor fluoroorotic acid (FOA). FOA is added to complete media (CMagar) at a concentration of 1 mg/ml. CM contains in grams per litre:malt extract, 20; glucose, 10; peptone, 1; agar, 15.

A. fumigatus spores are spread onto CM/FOA plates at a concentration of10⁷ and 10⁸ per plate. Plates are incubated at 37° C. until coloniesappear (usually 3-5 days). Colonies are then picked onto CM/FOA replicaplates for purity and to prepare for confirmation of the nutritionalstatus.

Confirmation of apyrG⁻ phenotype is achieved by checking the requirementfor uracil/uridine in the growth media. Two sets of minimal media (MM)are prepared, one supplemented with uracil/uridine (10 mM each finalconcentration) and one with no supplementation. MM contains in grams perlitre: NaNO₃, 6.0; KH₂PO₄, 1.52; KCl, 0.52; MgSO₄O.7H₂O, 0.52; glucose,10.0; trace element solution, 1.0 ml; media is adjusted to pH 6.5 withKOH. Trace element solution contains in grams per litre: FeSO₄, 1.0;ZnSO₄.7H₂O, 8.8; CuSO₄.5H₂O, 0.4; MnSO₄.4H₂O, 0.15; Na₂B₄O₇.10H₂O, 0.1;(NH₄)₆Mo₇O₂₄.4H₂O, 0.05. Uracil is quite insoluble and is made at 20 mMstock in water, uridine is made up at 500 mM stock in water. Bothsolutions are filter sterilised. CM and MM are made up and autoclaved insmall volumes in order to compensate for the large volume of uracilwhich needs to be added (due to its low solubility).

Suspected pyrG⁻ mutants are replica plated onto the two agars andincubated at 37° C. True pyrG⁻ mutants should emerge within 48 h onplates supplemented with uracil/uridine and no growth should occur onthe corresponding plate containing no supplementation.

1.3.3 Isolation of Haploid Double Auxotrophs (pyrG⁻/niaD⁻ andpvrG⁻/cnx⁻)

The isolation of haploid double auxotrophs (pyrG⁻/niaD⁻ and pyrG⁻/cnx⁻)is necessary for the production of diploids containing a doublepyrG⁻/pyrG⁻ (phenotype. The resulting pyrG⁻/pyrG⁻ diploids can then beused in transformation experiments utilising plasmids pRic3, ppyrG, pMB2and pMB3 all of which use the pyrG gene as a marker for DNA integration.

Using a haploidpyrG mutant (produced as in section 1.3.2) as theparental strain the exact protocol was followed as in section 1.3.1(Nitrate assimilation mutants (niaD⁻ and cnx⁻)). In this case all mediaused for the selection and confirmation of nitrate assimilation mutantswas supplemented with uracil/uridine (10 mM each final concentration)due to the presence of the pyrG⁻ phenotype.

The order of making the double auxotrophs was found to be important.Starting with a niaD⁻ or cnx⁻ mutant as the parental strain we wereunable to then isolate a niaD⁻/pyrG⁻ or cnx⁻/pyrG⁻ double mutant. Allour double mutants were made using a haploidpyrG⁻ mutant as the parentalstrain.

1.4 Production of Diploids in A. fumigatus:

These methods are based around a paper published by Strømnæs and Garberin 1963 (Strømnæs, Ø and Garber, E. D. (1963) Genetics 48, 653-662).Using the following methods diploid strains can be produced which areeither (niaD⁻/cnx⁻) when using single auxotrophic markers or(pyrG⁻/pyrG⁻/niaD⁻/cnx⁻) when using double auxotrophic markers. Diploidswith the niaD⁻/cnx⁻ phenotype are used in transformation experimentswhich utilise hygromycin resistance as a marker for DNA integration.Diploids with the pyrG⁻/pyrG⁻/niaD⁻/cnx⁻ phenotype are used intransformation experiments which utilise the pyrG gene as a marker forDNA integration.

1.4.1 Frontier Method

This is our method of choice for the production of diploids in A.fumigatus. Spores from niaD⁻ and cnx⁻ mutants, or niaD⁻/pyrG⁻ andcnx⁻/pyrG⁻ mutants (both at 10⁶ conidia per ml) were each inoculated onto one half of an agar plate containing Vogel's agar (Vogel, H. J.(1956) which is a convenient growth medium for Neurospora (medium N)Microbiol. Gen. Bull. 13: 42-44). Plates were incubated at 28° C. untilthe colonies had developed sufficiently to merge. Conidia were collectedfrom mycelia at the junction of the two colonies. Conidia were platedonto media containing nitrate (10 mM) as the sole nitrogen source andincubated at 37° C. until colonies had developed. Colonies growing atthis stage were regarded as presumptive diploids. The leading edge ofgrowing colonies were transferred to fresh nitrate media and allowed todevelop. Conidia from these colonies were then transferred to nitratemedia and incubated at 37° C.

During the initial step of this method the plates are incubated at 28°C. as opposed to 37° C. Incubation at the lower temperature slows downthe growth rate, increasing the chance of heterokaryon formation and thesubsequent production of diploids.

When double auxotrophs were used to produce diploids all media wassupplemented with uracil/uridine (10 mM each final conc) due to thepresence of the pyrG⁻ phenotype.

1.4.2: Mat Method

Conidia from both niaD⁻ 0 and cnx⁻ mutants, or niaD⁻/pyrG⁻ andcnx⁻/pyrG⁻ mutants were collected and counted. Fifty μl of conidia(approximately 1×10⁶ per ml) from each mutant phenotype was mixed with200 μl of complete medium (CM broth) and incubated overnight at 37° C.CM broth contains in grams per litre of water: malt extract, 20;bacto-peptone, 1; D-glucose, 20 (Rowlands & Turner. (1973) Molecular andGeneral Genetics 126: 201-216.). The resulting mycelial mat was placedon minimal media containing 10 mM nitrate as the sole nitrogen sourceand incubated at 37° C. until growth had occurred. Subcultures of anyoutgrowths of the primary inoculum were taken by removing sections ofthe leading edge and transferring to fresh nitrate medium. Growth onthis media indicates a stable diploid colony.

The ability to grow on nitrate media indicated the formation of adiploid colony since neither the niaD⁻ or cnx⁻ (nor niaD⁻/pyrG⁻ orcnx⁻/pyrG⁻) mutants can grown on this media. Therefore, any colonygrowing on nitrate must have a functional gene i.e. obtained throughgenetic fusion of the two mutants.

When double auxotrophs were used to produce diploids all media wassupplemented with uracil/uridine (10 mM each final conc) due to thepresence of the pyrG⁻ phenotype.

1.4.3: Protoplast Fusion

Conidia (10⁹ total) from niaD⁻ and cnx⁻ mutants, or niaD⁻/pyrG⁻ andcnx⁻/pyrG⁻ mutants were inoculated into 100 ml Vogel's medium andincubated for 16 h at 37° C. Germlings were harvested by filtration andsuspended in 50 ml protoplasting buffer, PB (1M NaCl, 10 mM CaCl)containing lysing enzyme at 2 mg/ml. Cultures were incubated at 30° C.with constant shaking (100 rpm) for 30-90 min. Protoplast formation wasmonitored microscopically until around 80-90% of germlings wereconverted to protoplasts. Protoplasts were harvested by filtration,concentrated by centrifugation (2000 g for 5 min) washed in PB andresuspended in sorbitol buffer (0.9M sorbitol, 0.125M EDTA pH 7.5).Protoplasts were diluted to a concentration of 10⁷ per ml. Equal amounts(100 μl) of protoplasts from both niaD⁻ and cnx⁻ mutants, or niaD⁻/pyrG⁻and cnx⁻/pyrG⁻ mutants were mixed with 100 μl of 30% PEG and placed onice for 20 min.

100 μl and 50 μl aliquots were added to tempered nitrate media (10 mMnitrate final) supplemented with 1.2M sorbitol and 10 mM CaCl. Plateswere incubated at 30° C. until colonies emerged. Colonies weretransferred to fresh nitrate medium and allowed to develop.

When double auxotrophs were used to produce diploids all media wassupplemented with uracil/uridine (10 mM each final concentration) due tothe prescience of the pyrG⁻ phenotype.

1.5 Production of Diploids in C. glabrata:

C. glabrata is naturally haploid but diploid strains may be produced asfollows.

Suitable parental C. glabrata haploid strains are selected which havelost the ability to make specific metabolites or enzymes. Parentalstrains may be selected by screening for natural spontaneous mutationsor by inducing mutation through UV mutagenesis or treatment withchemical mutagens e.g. nitric acid.

Where spontaneous mutations are used, the desired mutations are screenedby plating liquid cultures of C. glabrata onto solid complete mediawhich may contain vitamin and mineral supplements to allow for growth ofdesired mutant phenotypes.

Colonies are then replica plated on to minimal media which are deficientin one particular nutrient only. Mutant auxotrophs can then beidentified by comparing their is growth on both complete and minimalmedia. Organisms which fail to grow on minimal media but grow onsupplemented complete media are classified as auxtrophic for thedeficient compound. It is preferable that one parent is auxotrophic fortwo compounds preferably from uracil, arginine, leucine, histidine andadenine and that the other parent is also auxotrophic for two of thesaid compounds but its auxotrophy differs from the other parent.

Formation of diploid C. glabrata may be achieved by joint culture of twoparental auxotrophs in liquid media containing the necessary nutrientsfor both parents to grow. This is followed by sub-culturing organismsfrom this media to minimal media deficient in the auxotrophic nutrientsof either parent. Diploid strains can then be isolated from this media.

Additionally diploid strains may be formed from haploid parentalauxotrophs by polyethylene glycol (PEG) mediated protoplast fusion.Protoplasts from each parental haploid auxotroph are formed by digestionof the cell wall with a suitable commercial protoplasting enzyme (thepreferred enzyme being Zymolase). Protoplasts from each haploid parentare mixed (the joint culture stage) in the presence of PEG. Protoplastsare then plated onto solid minimal medium deficient in auxotrophicnutrients of the parent haploid strains but supplemented with an osmoticstabiliser to prevent protoplast bursting. Prototrophic colonies thatderive from this minimal media are presumptive diploids.

1.6 Re-Haploidisation

The following method was used for the re-haploidisation of diploidcolonies of C. glabrata and A. fumigatus. Diploid colonies weresubjected to re-haploidisation by the use of the mitotic inhibitorfluorophenylalanine (FPA).

Conidia (A. fumigatus) or cells (C. glabrata) were collected from stablediploid colonies and spread plated onto complete media containingnitrate and 0.01-0.2% FPA and incubated at 37° C. for 3 days or untilrapidly growing sectors emerged (A. fumigatus). Conidia were collectedfrom each sector (A. fumigatus) or colonies picked (C. glabrata) andplated onto nitrate, nitrite and hypoxanthine media and the nitrogenutilisation profiles of the resulting conidia (A. fumigatus) or cells(C. glabrata) assessed. Colonies with the nitrogen utilisation profilesof the parental strains could then be re-isolated indicating a haploid.

1.7 Transformation Experiments:

1.7.1 Electroporation

This is our method of choice for the production of transformants in A.fumigatus.

Approximately 125 ml of YG (0.5% (w/v) yeast extract, 2% (w/v) glucose,5 mM each uridine and uracil) medium was inoculated with 109 conidiafrom an auxotrophic haploid or diploid strain. The preferred auxotrophicstrains were derived from AF300 or AF293.

For transformation of swollen conidia, cultures were incubated for 4 hat 37° C. with constant shaking (200 rpm). For transformation ofgermlings, incubation was for 8 h again at 37° C. Swollen spores orgermlings were collected by centrifugation (5 min at 5000×g) and washedin 200 ml of ice cold sterile water. Spores or germlings wereresuspended in 10-12 ml of YED (1% (w/v) yeast extract, 1% (w/v)glucose, 20 mM HEPES, pH 8.0), and incubated at 30° C. for a further 60min. Spores/germlings were collected by centrifugation as described andresuspended in 1 ml of EB buffer (10 mM Tris pH 7.5, 270 mM sucrose, 1mM lithium acetate).

Electroporation was carried out in 50 μl aliquots of spores/germlings(5×10⁷ germlings/conidia). 50 μl of swollen conidia/germlings weretransferred to an electroporation cuvette, on ice, and 1-5 μg oftransforming DNA added. This may be in the form of circular orlinearised plasmid ppyrG, pMB2, pMB3 or another plasmid or DNA speciescarrying a complimentary DNA sequence to the auxotrophic markers of therecipient strain.

Conidia/germlings and transforming DNA were mixed and left on ice for 30min. Conidia/germlings were electroporated in a Gene Pulser IIinstrument (Bio-Rad Ltd) set at 1 kV, 400 Ω and 25 ΞF.

1 ml of cold YED was added to the cuvette and incubated at 37° C. for 1h. Aliquots were spread on non-selective agar (Vogel's, minimal media orcomplete media) without urine or uracil. Colonies growing onnon-selective media were deemed putative transformants.

1.7.2 PEG-Mediated Protoplast Transformation

The following method is used for transformation of protoplasts of C.glabrata which are produced as described in section 1.5, and A.fumigatus as herein described below.

Conidia (A. fumigatus) from AF293 pyrG strains with additional niaD⁻ orcnx⁻ auxotrophy were cultured on solid CM media supplemented with 10 mMuracil and 10 mM uridine at 37° C. for 2-4 days. Conidia were collectedin 10 ml PBS/0.1% (w/v) Tween 80 and counted using a heamocytometer.1×10⁷ conidia were inoculated into 50 ml Vogel's media in Erlinmeyerflasks, again supplemented with 10 mM uracil and 10 mM uridine. Flaskswere incubated overnight at 37° C. with constant shaking and theresultant mycelia was harvested by vacuum filtration. Mycelia wasresuspended in 20 ml protoplasting buffer (1M NaCl, 10 mM MgCl₂) andprotoplasting enzyme (zymolase) added to a final concentration of 1-5mg/ml. This mycelia/enzyme suspension was incubated at 30° C. withgentle shaking (80 rpm) and protoplast generation followedmicroscopically over a maximum 3 h period. Protoplasts were harvestedwhen most of the mycelia had been converted to protoplasts by filtrationthrough sterile gauze.

Protoplasts were pelleted by gentle centrifugation (800 g×5 min) andwashed 2×10 ml in protoplasting buffer before finally being resuspendedin stabili sing buffer (0.9 M sorbitol, 0.1 M EDTA) to a finalconcentration of 2×10⁷ protoplasts/ml. Up to 2 μg of linearisedtransforming plasmid (either ppyrG, pMB2 or pMB3) was added to theprotoplast suspension and placed on ice for 30 min.

500 μl PEG 3000 (40% (w/v)) was added dropwise to 500 μlprotoplast/plasmid suspension containing 10⁷ protoplasts. This PEGprotoplast suspension was incubated on ice for a further 15 min beforecentrifugation at 100 g for 10 min. The supernatant was removed andreplaced with 500 μl of stabilising buffer. Transformation reactionswere either plated onto the surface of Vogel's agar plates containing 1%(w/v) glucose, 1.2 M sorbitol or mixed with 20 ml of molten tempered (to40-45° C.) of the same agar.

Plates were incubated at 37° C. for up to 14 days and inspected forgrowth. Colonies growing on this selection media were deemed putativetransformants.

1.7.3 Agrobacterium-Mediated Transformation

The A. tumefaciens strains containing the vectors pRic1, pRic2 or pRic3were grown at 29° C. overnight on LB plates containing: rifampicin,kanamycin, ampicillin and gentamicin for A. tumefaciens GV3101containing pRic2 or pRic3; rifampicin, kanamycin and gentamicin for A.tumefaciens GV3101 containing pRic1; rifampicin and kanamycin for A.tumefaciens LBA4404 containing pRic1; rifampicin, kanamycin andampicillin for A. tumefaciens LBA4404 containing pRic2 or pRic3. Allantibiotic concentrations are as stated in the Plasmid constructionsection (1.2.3) except for gentamicin at 20 μg/ml. A single colony wasstreaked on a minimal medium plate. Minimal medium (MM) contains ingrams per litre: K₂HPO₄, 2.05; KH₂PO₄, 1.45; NaCl, 0.15; MgSO₄.7H₂O,0.50; CaCl.6H₂O, 0.1; FeSO₄.7H₂O, 0.0025; (NH₄)₂SO₄, 0.5; glucose, 2.0.The plates were incubated at 29° C. for 1 to 2 days.

Several colonies were inoculated in minimal medium containing theappropriate antibiotics and grown at 29° C. overnight. After dilution ofA. tumefaciens cells to an OD_(660nm) of approx. 0.15 in inductionmedium the culture was grown for 6-7 hours at 29° C. The inductionmedium (1M) differs from MM in that the 2 grams per litre glucose wasreplaced by 10 mM glucose and 40 mM MES (pH 5.3), 0.5% glycerol (w/v)and 200 μM acetosyringone (AS) were added. In order to confirm that thetransformation of A. fumigatus by A. tumefaciens is dependent on T-DNAtransfer, a negative control was included in which the vir inducer ASwas omitted.

Conidia were obtained by growing the A. fumigatus strains (haploid ordiploid) at 37° C. on Vogel's minimal medium agar plates for severaldays and subsequently washing the surface of the plates withphysiological salt solution and then filtering the conidial suspensionthrough glass wool.

For transformation of conidia, conidia were diluted in physiologicalsalt solution at a concentration of 10⁶ or 10⁷ conidia per ml and 100 μlwas mixed with 100 μl of the A. tumefaciens culture (induced as detailedusing IM). Subsequently, the mixtures were plated on nitrocellulosefilters placed on absorbent pads containing IM (reduced glucoseconcentration to 5 mM) and incubated at 25° C. for 2 to 3 days. Thenegative control samples were incubated on IM pads in which the virinducer AS was omitted. After this incubation period, the filters weretransferred to Vogel's medium agar plates containing cefotaxime (200 μM)to kill the A. tumefaciens cells and hygromycin (400 μg/ml) to selectfor fungal transformants.

1.7.4 Early Identification of Transformants

Using the previously described transformation methods it was foundbeneficial to be able to identify transformants at the earliest possiblestage of their development. Putative transformants were identified usinga stereo microscope (Zeiss Ltd) when they were not yet visible to thenaked eye and were picked using a sterile, fine gauge needle andtransferred to individual petri dishes containing selective agar.

1.8 DNA Isolation, PCR and Southern Analysis:

To obtain mycelial material for genomic DNA isolation, approximately 10⁷A. fumigatus conidia were inoculated in 50 ml of Vogel's minimal mediumand incubated with shaking at 200 rpm until late exponential phase(18-24 h) at 37° C. The mycelium was dried down onto Whatmann 54 paperusing a Buckner funnel and a side-arm flask attached to a vacuum pumpand washed with 0.6 M MgSO₄. At this point it is possible to freeze-drythe mycelium for extraction at a later date. The mycelium (fresh orfreeze dried) was ground to a powder using liquid nitrogen in a −20° C.cooled mortar. The powder was added to a 1.5 ml microcentrifuge tubeusing an ethanol-cleaned spatula (no more than 0.4 ml), 0.6 ml ofextraction buffer (0.7 M NaCl; 0.1 M Na₂SO₃; 0.1 M Tris-HCl pH 7.5; 0.05M EDTA; 1%(w/v) SDS) heated to 65° C. was added and the microfuge tubewas incubated at 65° C. for 20 min. 0.6 ml of chloroform/isoamyl alcohol(24:1) was added, the tube was vortex mixed thoroughly and incubated onice for 30 min. The tube was centrifuged at 12,000×g for 30 min and theaqueous phase carefully transferred to a fresh microfuge tube withoutdisturbing the interface. An equal volume of isopropanol was added,mixed by inversion and incubated at room temperature for 10 minutes. Thetube was centrifuged at 2000×g for 5 min, the supernatant was removedand the pellet allowed to air dry. The pellet was suspended in 200 μl of18 MΩ water and incubated at 37° C. for 15-30 min. 100 μl of 7.5 Mammonium acetate was added, mixed by inversion and incubated on ice for1 hour. The tube was centrifuged at 12000×g for 30 min, the supernatanttransferred to a fresh tube and 0.54 volumes of isopropanol were added,mixed by inversion and incubated at room temperature for 10 minutes. Thetube was centrifuged at high speed for 5 min, the supernatant wasremoved and the pellet washed in 500 μl of 70% ethanol. The tube wascentrifuged at high speed for 5 min and all the ethanol was removed. Thepellet was air dried and suspended in 100 μl of TE (10 mM Tris-HCl pH7.5; 1 mM EDTA) or 18 MΩ water. The DNA was treated with RNase A (1 μlof 1 mg/ml stock) before use.

To confirm the transformation of A. fumigatus with plasmid DNAcontaining the pyrG gene (ppyrG, pRic3, pMB2 and pMB3) we subjected thepurified DNA from transformed fungal colonies to PCR and southernanalysis.

PCR was carried out using the following primers:

pyrG1: 5′-gca gag cga ggt atg tag gc-3′; (Seq ID No 20) pyrG2: 5′-aagccc tcc cgt atc gta gt-3′; (Seq ID No 21) pyrG3: 5′-ata cct gtc cgc ctttct cc-3′; (Seq ID No 22) pyrG4: 5′-ttt atc cgc ctc cat cca-3′; and (SeqID No 23) pyrG5: 5′-gcc ttc ctg ttt ttg ctc ac-3′. (Seq ID No 24)

All the pyrG primers (pyrG1-pyrG5) were designed to pUC19 sequence whichis present in the pyrG transformation cassettes. Designing primers tothe actual pyrG gene would be of no diagnostic use as pryG⁻ strainsstill carry the pyrG sequence.

To confirm the transformation of A. fumigatus with T-DNA containing thehph gene from A. tumefaciens we subjected the purified DNA fromtransformed fungal colonies to PCR and southern analysis.

hph6: (Seq ID No 1) 5′-cga tgt agg agg gcg tgg at-3′; hph7: (Seq ID No2) 5′-atc gcc tcg ctc cag tca at-3′; hph12: (Seq ID No 3) 5′-ctt agc cagacg agc ggg tt-3′; hph13: (Seq ID No 4) 5′-caa gac ctg cct gaa accga-3′; and hph14: (Seq ID No 5) 5′-tcg tcc atc aca gtt tgc ca-3′.

For southern analysis, approximately 2.5 μg DNA was digested with arestriction enzyme which does not cut within the inserted DNA sequencefor 16 hours and separated on a 0.8% agarose TAE gel. DNA wastransferred to a Hybond N membrane by capillary blotting (overnight) andthe membrane was pre-hybridized according to the Hybond protocol. Probesspecific for the pyrG or hph gene were digoxigenin (DIG) (Feinberg, A.P. and Vogelstein, B. (1983) Analytical Biochemistry 132, 6-13) orα³²P-labelled PCR products amplified using the primers detailed in thissection.

1.9 Isolation of Mutated Gene Sequences

1.9.1 TAIL-PCR

As the T-DNA region of pRic1 does not contain a bacterial origin ofreplication, plasmid rescue (see section 1.9.2) cannot be used toisolate the mutated gene of interest. In this case we used a methoddescribed as thermal asymmetric interlaced PCR (TAIL-PCR: Liu et al.(1995) The Plant Journal 8: 457-463).

Primers were designed to the NPTII gene region of pRic1. This region ofDNA is inserted into the mutated gene during transformation and henceacts as a marker for the mutated gene.

The primers used were:

NPT1: (Seq ID No 6) 5′-tcc cgc tca gaa gaa ctc gtc aa-3′; NPT2: (Seq IDNo 7) 5′-ttg ggt gga gag gct att cgg ct-3′; NPT3: (Seq ID No 8) 5′-tgttgt gcc cag tca tag ccg aa-3′; NPT4: (Seq ID No 9) 5′-agc cga ata gcctct cca ccc aa-3′; NPT5: (Seq ID No 10) 5′-cag att att tgg att gag agtga-3′; AD1: (Seq ID No 11) 5′-ntc ga(g/c) t(a/t)t (g/c)g(a/t) gtt-3′;AD2: (Seq ID No 12) 5′-ngt cga (g/c)(a/t)g ana (a/t)ga a-3′; and AD3:(Seq ID No 13) 5′-(a/t)gt gna g(a/t)a nca nag a-3′.

-   -   where n=any base, (x/y)=wobble position.

NPT1 and NPT2 were used to check that the NPTII sequence is present inthe transformants. NPT1-NPT3 are nested primers. NPT3 is used in theprimary TAIL-PCR, NPT4 in the secondary TAIL-PCR and NPT5 in thetertiary TAIL-PCR. AD1-AD3 are arbitrary degenerate primers. TAIL-PCRcycle settings were as described in the published method (Liu et al.supra). PCR fragments isolated from the tertiary TAIL-PCR by this methodwere cloned and sequenced by conventional molecular biologicaltechniques.

1.9.2 Plasmid Rescue

As the T-DNA region of pRic2 and pRic3 and plasmids ppyrG, pMB2 and pMB3all contain a bacterial origin of replication, the technique of plasmidrescue can be used to isolate the mutated gene of interest. Genomic DNAisolated from transformed A. fumigatus (section 1.8) was digested tocompletion with a restriction enzyme that does not cut within theinserted DNA. This digested DNA was then purified and re-ligated with T4DNA ligase. The random sized, closed circular DNA molecules resultingfrom this process were then used to transform E. coli strain Top10 byelectroporation using a Biorad Genepulser (2.1 kV, 200 Ω, 25 μF).Transformed cells were plated on LB agar plates containing ampicillin(100 μg/ml). These plates were incubated at 37° C. until single coloniesstarted to form (these single colonies represent E. coli Top10containing a plasmid with a bacterial origin of replication i.e. fromthe inserted DNA.). DNA was isolated from these cells and sequenced byconventional molecular biological techniques.

1.9.3 Inverse PCR

In a similar manner to plasmid rescue, genomic DNA isolated fromtransformed A. fumigatus was digested to completion with a restrictionenzyme that does not cut within the inserted DNA. This digested DNA wasthen purified and re-ligated with T4 DNA ligase. Instead of beingtransformed into E. coli as in the plasmid rescue method, the randomsized, closed circular DNA molecules resulting from this process weresubjected directly to PCR.

This technique may be used when trying to isolate the mutated gene fromtransformants produced using linearised plasmids which have been cutwith a known restriction enzyme. For example, we have designed primersto isolate the mutated gene of interest from transformants producedusing pMB3 linearised with XbaI.

PCR was carried out using the following primers:

RCpyrG5: (Seq ID No 14) 5′-gtt tga ggc gaa ttc tc-3′; RCpyrG3: (Seq IDNo 15) 5′-ctc tca gac aga att cgt-3′; pMB3R: (Seq ID No 16) 5′-atc catcac act ggc g-3′; T7pro: (Seq ID No 17) 5′-taa tac gac tca cta taggg-3′; M13-20: (Seq ID No 18) 5′-gta aaa cga cgg cca g-3′; and M13-40(Seq ID No 19) 5′-gtt ttc cca gtc acg ac-3′.1.10 Identification of Mutated Genes

All DNA sequencing was carried out by external contract (MWG Biotech UK10 Ltd., Waterside House, Peartree Bridge, Milton Keynes, MK6 3BY).Sequence data obtained was compared to sequences in the public domaindatabases via BLAST searches (National Centre for BiotechnologyInformation.

2. Results

2.1 Haploid Transport Library

Using the protocols detailed above, the inventors transformed haploidAF300 and AF293 with A. tumefaciens (LBA4404 and GV3101) and haveapproximately 300 (MA1-MA300) transformants frozen down (−80° C.) whichexhibit growth (at various radial growth rates) on hygromycin (400 μg/mlor higher). One of these transformants (MA205) was taken complete circleto show that A. fumigatus could be transformed using theAgrobacterium-mediated method and that T-DNA could be integrated at asingle gene locus and that locus could be identified by DNA sequencingand homology to known genes examined by the use of bioinformatics.

A. fumigatus transformant MA205 was produced by the transformation ofAF300 with A. tumefaciens GV3101 containing pRic1. PCR with primers hph6and hph7 revealed that the hygromycin gene was present in the genomicDNA. Because pRic1 does not contain a bacterial origin of replication(this origin is present in pRic2 and pRic3 by the introduction of pUC 18DNA into the HindIII site (see section 1.2.2)) which allows theisolation of genic DNA sequences (disrupted gene etc) flanking theinserted T-DNA by plasmid rescue, we used a flanking region isolationmethod known.

As TAIL PCR (see section 1.9.1). The DNA fragment isolated by thismethod was sequenced by MWG Biotech UK Ltd. (see SEQ ID No. 27) and wastranslated in all six reading frames to yield a partial protein sequenceof 234 amino acids. Using BLAST (National Centre for BiotechnologyInformation this 5 protein sequence showed strongest sequence homology(41% at the amino acid level) to a hypothetical 35.1 KD protein in theNAMS-GARI intergenic region of Saccharomyces cerevisiae (sp/P38805/YH08YEAST and see FIG. 12).

Transformation of haploid AF293 using the preferred electroporationmethod has yielded transformants which are pyrG⁺ by their growth on nonselective media (see section 1.7.1) and which have been confirmed astrue transformants by diagnostic PCR (see section 1.8). Southern blotsof some of these transformants indicating the presence of random, singleand/or multiple insertion events is shown in FIG. 13 (lanes A-F).

2.2 Diploid General Library:

We have produced AF293 and AF300 diploids using the above-describedmethods (see section 1.4) and have shown that these are in fact true A.fumigatus diploids via re-haploidisation with the use of the mitoticinhibitor fluorophenylalanine (FPA) (see section 1.6).

These diploids were used to form mutant banks according to the presentinvention utilising the transformation methods detailed in section 1.7.Diploid transformants have been isolated which are pyrG⁺ by their growthon non selective media and which have been confirmed as truetransformants by diagnostic PCR. A southern blot showing an example of asingle integration event in a diploid transformant is shown in FIG. 13(lane G).

1. A method of selecting a microorganism clone with a mutation in anessential gene comprising: (i) providing a mutant bank of diploidmicroorganism cells comprising a population of diploid microorganismcells in which mutations have been randomly induced, wherein at leastone cell has a random mutation which is lethal to the haploid form ofthe cell; (ii) exposing the diploid microorganisms to an agent thatinduces the microorganisms into haploid form; and (iii) selecting adiploid clone for which the haploid microorganism is not viable, thenon-viability indicating that the selected clone has a mutation in anessential gene, wherein the microorganism is Aspergillus fumigatus. 2.The method according to claim 1 which further comprises identifying themutated gene in the selected microorganism clone.
 3. The methodaccording to claim 1, wherein in the mutant bank a plurality of cells inthe population each individually have a mutation which disrupts theactivity of at least one gene, so that collectively the said pluralityof cells have mutations in a plurality of genes within the genome. 4.The method according to claim 3, wherein the said plurality of genesmakes up 10% of the genes within the genome.
 5. The method according toclaim 3, wherein the said plurality of genes makes up all the genes ofthe genome of the microorganism.
 6. The method according to claim 1,wherein the mutant bank is formed by an insertional mutagenesis method,wherein a DNA molecule is inserted into each gene to cause the mutation.7. The method according to claim 6, wherein the DNA molecule is selectedfrom the group consisting of a selectable marker, a pyrG gene, a pyrGgene from A. fumigatus, a pyrG gene harboured on a plasmid and anAspergillus pyrG gene harboured on a plasmid.
 8. A method of generatinga mutant bank of microorganisms comprising a population of mutant A.fumigatus cells in which mutations have been randomly induced, whereinat least one cell has a random mutation that disrupts the activity of atleast one gene, said method comprising the steps of: (i) culturing apopulation of A. fumigatus cells; and (ii) inducing a random mutation inat least one cell of the population which mutation disrupts the activityof at least one gene in the genome of the cell, said cells being diploidand being inducible into haploid form.
 9. A method according to claim 8,wherein the mutant bank is formed by an insertional mutagenesis method,wherein a DNA molecule is inserted into each gene to cause the mutation.10. A method according to claim 9, wherein the DNA molecule is selectedfrom the group consisting of a selectable marker, a pyrG gene, a pyrGgene from A. fumigatus, a pyrG gene harboured on a plasmid and anAspergillus pyrG gene harboured on a plasmid.
 11. A method according toclaim 9, wherein the DNA is introduced into the microorganism by amethod selected from the group consisting of electroporation, protoplasttransformation, PEG-mediated transformation and Agrobacterium-mediatedtransformation.
 12. A method according to claim 8, wherein the diploidmicroorganism is a mutant for the pyrG phenotype.
 13. A method accordingto claim 12, wherein the diploid mutant microorganism is generated usingthe following steps: (i) isolating a first haploid mutant comprising afirst auxotrophic marker wherein the first auxotrophic marker results inthe pyrG⁻ phenotype; (ii) isolating a second haploid mutant using thefirst haploid mutant isolated in step (i), said second mutant comprisingthe first pyrG⁻ auxotrophic marker and a second auxotrophic marker;(iii) isolating a third haploid mutant using the first haploid mutantisolated in step (i), said third mutant comprising the first pyrG⁻auxotrophic marker and a third auxotrophic marker; and (iv) mating thesecond and third haploid mutants isolated in steps (ii) and (iii) togenerate the diploid mutant exhibiting the pyrG⁻ phenotype.
 14. A methodaccording to claim 13, wherein the second and third auxotrophic markersare selected from niaD⁻ or cnx⁻.
 15. A method according to claim 13,wherein step (iv) of the method comprises use of the Frontier method,Mat method or protoplast fusion for the production of diploidmicroorganisms in A. fumigatus.
 16. A method according to claim 13,wherein the haploid mutants are incubated at a temperature in a rangeselected from the group consisting of 20 to 36° C., 24 to 33° C. and 26to 30° C.
 17. A mutant bank of diploid microorganisms comprising apopulation of mutant cells in which each individual cell has a randommutation that disrupts the activity of one gene, said populationcollectively having a mutation in every gene within the genome andwherein the mutant bank may be induced into haploid form, wherein themicroorganism is A. fumigatus.